The gameplay remains similar to the prequel however there have been a few changes made to each species:
The Predator now has the ability to recharge his own energy.
The Alien can now destroy things.
The Marine has gained a few new weapons, along with the ability to don an exo-suit ("Alice") for one level.
Almost half a century after the events of Aliens and Alien³, the Weyland Yutani Corporation followed the flight telemetry of the derelict spacecraft found on LV-426 and discovered a world full of life and dotted with the ruins of an extraterrestrial civilization—and infested with Xenomorphs. This planet would become known as LV-1201.
The Company establishes a long-term research facility under Dr. Eisenberg to study the planet, the ruins, and the aliens. The facility consists of a Primary Operations Complex and a network of five massive Forward Observation Pods suspended over a canyon.
- Corporal Andrew "Frosty" Harrison is the main human character in the game. A USCM Marine, he is not given much background, but is characterized by a refusal to abandon his fellow marines. Other than combat, Harrison specializes in electronics and is also qualified to pilot an elite "Exo-Suit". It is also mentioned in the game by other marines that Harrison is nicknamed 'Frosty' for being 'cool' in very hot engagements (e.g. attempting a solo rescue while being chased by a large swarm of Xenomorphs). It is also suggested that Frosty has had poor luck with women, characterized when trying to hit on Dunya and Tomiko.
- A Predator is the primary Predator character in the game. 19 years before, he and his clan attacked a Weyland-Yutani outpost, where he chased and downed a Colonial Marine known as Rykov. Now, knowing Rykov is still alive, the Predator has come to LV-1201 to finish the job. It is implied he is a high-ranking hunter (speculated by Rykov to be a Prince) by the conversations of human scientists studying his mask after he is captured.
- An Alien drone is the central Xenomorph character in the game. A both intelligent and aggressive drone, it caused the Incident in attempting to escape from the Primary Operations Complex back to the hive. it also rescued the queen, captured Eisenberg, saved the hive from being destroyed, and killed multiple Predators (3 necessary, 1 optional).
- Dr. Eisenberg is the head researcher in charge of the LV-1201 facility. He was the only survivor of a disastrous first expedition; when a massive storm cut off his outpost's security systems and communications, he was forced to kill and eat his colleagues. He suffered severe acid burns during a rescue attempt; as the game progresses, it is revealed that he only "survived" because his father, a major figure in the synthetic human research industry, was able to port his consciousness into an identical android body. Because of that event, Eisenberg harbors a deep hatred—and a paralyzing fear—of Xenomorphs. He is captured by the playable alien character.
- General Vasili Rykov is the head of the Iron Bears or "Corporates", a group of mercenaries hired by Weyland-Yutani to assist operations on LV-1201. Years ago, he was a colonial marine stationed at a Weyland-Yutani bio-weapons facility on the planet Korari when it was attacked by the same Predator whom the player controls. Rykov was severely injured; he was paralyzed until he underwent an experimental procedure which replaced his spinal column with its synthetic equivalent. The treatment was only partially successful; Rykov must take powerful black-market narcotics in order to function. Rykov has since become obsessed with wiping out the Predator race. In an exo-suit, he fights the playable predator character but ultimately dies. Rykov has a strong Russian accent.
- The Empress is the name given to the primary Xenomorph Queen by Eisenberg. He wants to capture and control her.
- Major McCain is the commanding officer of the Marines dispatched to LV-1201 under Weyland-Yutani's request. He is a fine leader, but is oblivious to what Rykov and Eisenberg have in store for him and his men.
- Duke is a marine (seemingly of Latino descent) under the command of Major McCain. Both sarcastic and relatively cowardly, he is considered an annoyance by the other marines (easily comparable to Hudson from Aliens). He is Corporal Harrison's friend.
- Johnson is the premier dropship pilot aboard the Verloc. She carries out the marines' dropship missions, and rescues Harrison towards the end of the Marine campaign. She has a strong British accent and a strong-willed personality, as shown in her response to Major McCain's question, "Where the hell did you learn to fly?"; "Flight school! The same place that taught me not to fly blind in hurricane winds!"
- Decker is a marine private in Sergeant Hall's team. He is badly burnt in an alien ambush and is put in an APC to heal. He heals quickly, however, and is later seen guarding McCain and the APCs. He is killed by the Alien Queen as Harrison enters their lair.
- Jones is an African-American marine under the command of Major McCain. A heavy weapons specialist.
- Sergeant Hall is a female African-American marine. She is second-in-command to Major McCain. During the raid on the POC, her squad is ambushed and she is captured and impregnated by the Xenomorphs. She dies when Harrison finds her, a chestburster exiting her stomach.
- Sergeant Blackwell is a marine under the command of Major McCain. He is head of Harrison's squad until they were separated. He and Shugi retreat back to McCain and guard him for the rest of the game. His survival varies: Sometimes, you can find his and Shugi's corpses in the Alien Queen's lair.
- Shugi is a marine under the command of Major McCain. He is part of Harrison's squad until they were separated. He and Blackwell retreat to Major McCain and stay in a marine APC or Dropship for the majority of the game. His survival varies: Sometimes, you can find his and Blackwell's corpses in the Alien Queen's lair.
- Tomiko is said to be an assemblyman's daughter who signed up for an internship on LV-1201 to find out what had happened to her brother, Kenji, who she suspects was murdered. She blackmails Harrison into finding a disk with evidence on it and later helps him out. She willingly sacrifices her life at the end of the Marine campaign to ensure the total destruction of the site and, with it, the genetic experimentation and research that the corporation had been conducting. However, the cryptic end message (see "Final Cutscene" below) suggests that her story might actually be bogus, and that she and her brother are in fact real corporate spies.
- Captain Richter is the captain of the cargo vessel Aurora and often ferries questionable objects (such as Alien eggs) with no questions asked for Weyland Yutani, though he often scans them to get an idea of what Weyland Yutani is doing. Presumably for such meddling, he is escorted by a security guard named Lloyd to Eisenberg, who has him tortured. Richter is killed when Tomiko destroys the Pods.
- Sergeant Cisco is a temperamental guard Sergeant. He gets angry at soldiers easily and has no friends in the Pods. He is killed when the Alien player facehugs him, and then bursts from his chest.
- Lloyd is the security guard who escorts Richter to Eisenberg. He is later seen complaining about being chewed out by Cisco and mentioning that "he has no friends".
- Stiles is a technician who assists Harrison while he is in the Pods (before Dunya betrays him). He is easily recognized because of his goatee. He is probably killed when Tomiko causes a meltdown in the Pods.
- Major Dunya is a Russian member of the Iron Bears. When Corporal Harrison disabled the security network in the tunnels connecting the POC to the Pods, he inadvertently disabled the security at the Pods themselves as well. Dunya's fiancé Dimitri was killed when Harrison shut off the security grid that was connecting the POC to the Pods and the Xenomorphs swarmed in and attacked. As a result, Dunya blamed Harrison for his death.
- Ivan is General Rykov's right hand man, who sees to it that all of the general's orders are followed. He is killed by Andrew Harrison while trying to prevent Harrison from escaping the Pods.
Note: Strangely, Ivan is encountered in Alien campaign at a point when he's supposed to be already dead. It is believed that the designers simply re-used Ivan's skin for another Iron Bear just as they did with Dimitri's.
- Chekov is one of General Rykov's henchmen. While returning from escorting his superior to the Hive, he is killed by The Alien.
- Popov is another henchman. He is killed by Aliens while escorting a Predator Artifact through the Pods.
- Serge is a Russian Iron Bear. He is killed by Aliens while retrieving the Artifact.
- Pamela is a doctor working in the pods. She is found by a rogue Predalien.
- Decker is an Iron Bear who has the same name as the marine, though there is no relation. A Texan, he does not agree with some of Rykov's tactics. He is killed by Aliens when retrieving the Artifact.
- Dimitri is a Russian foot soldier in the Iron Bears and is Dunya's fiancé. He is killed when out guarding a scientist outside the pod when Harrison shuts off the security grid. He makes his appearance in the Alien single-player campaign when he's guarding a technician who is fixing a generator powering an electrical laser fence, and getting swarmed by a bunch of Xenomorphs, which leads to his horrible death. He's also a playable character for the Corporate team in multiplayer.